Hydrogen-deuterium exchange mass spectrometry reveals the interaction of Fenna-Matthews-Olson protein and chlorosome CsmA protein

In green-sulfur bacterial photosynthesis, excitation energy absorbed by a peripheral antenna structure known as the chlorosome is sequentially transferred through a baseplate protein to the Fenna-Matthews-Olson (FMO) antenna protein and into the reaction center, which is embedded in the cytoplasmic membrane. The molecular details of the optimized photosystem architecture required for efficient energy transfer are only partially understood. We address here the question of how the baseplate interacts with the FMO protein by applying hydrogen/deuterium exchange coupled with enzymatic digestion and mass spectrometry analysis to reveal the binding interface of the FMO antenna protein and the CsmA baseplate protein. Several regions on the FMO protein, represented by peptides consisting of 123-129, 140-149, 150-162, 191-208, and 224-232, show significant decreases of deuterium uptake after CsmA binding. The results indicate that the CsmA protein interacts with the Bchl a #1 side of the FMO protein. A global picture including peptide-level details for the architecture of the photosystem from green-sulfur bacteria can now be drawn.     

A reconstituted light-harvesting complex from the green sulfur bacterium Chlorobium tepidum containing CsmA and bacteriochlorophyll a

Green sulfur bacteria possess two light-harvesting antenna systems, the chlorosome and the Fenna-Matthews-Olson (FMO) protein. In addition to self-aggregated bacteriochlorophyll (BChl) c, chlorosomes of Chlorobium tepidum contain a small amount of BChl a (ratio 100:1). The chlorosomal BChl a is associated with CsmA, a 6.2 kDa protein that accounts for more than 50% of the protein content of chlorosomes. This CsmA-BChl a complex is located in the chlorosome baseplate with the hydrophilic C-terminal part of CsmA in contact with the FMO protein. CsmA was purified from Chl. tepidum. Isolated chlorosomes were lyophilized and extracted with chloroform/methanol (1:1, v/v). The extract was further purified using gel filtration and reverse-phase HPLC and the purity of the preparation confirmed by SDS-PAGE. Mass spectrometric analysis showed an m/z of 6154.8, in agreement with the calculated mass of the csmA gene product after C-terminal processing. CD spectroscopy of the isolated protein showed that the main structural motif was an alpha-helix. We have reconstituted the isolated CsmA protein with BChl a in micelles of n-octyl beta-d-glucopyranoside. The resulting preparation reproduced the spectral characteristics of the CsmA-BChl a complex present in the chlorosome baseplate.     

Presence and significance of minor antenna components in the energy transfer sequence of the green photosynthetic bacterium Chloroflexus aurantiacus

Antenna components in the energy transfer processes of a green photosynthetic bacterium Chloroflexus aurantiacus were spectrally investigated by time-resolved fluorescence spectroscopy at −196°C on intact cells. Besides major antenna components so far reported, three minor components were resolved; those were Bchl c located at 785 nm, the baseplate Bchl a at 819 nm and Bchl a in the B808-866 complex at 910 nm. The last component was assigned to a longer wavelength antenna closely associated with a reaction center. An additional Bchl c fluorescence component was kinetically suggested to be present, which can be an energy donor to a major Bchl c. Presence of these minor components was signified in terms of (1) increase in the spectral overlap integral and (2) adjustment of the direction of dipole moments in the energy transfer sequence of intact cells.     

Computational determination of the pigment binding motif in the chlorosome protein a of green sulfur bacteria

We present a molecular-scale model of Bacteriochlorophyll a (BChl a) binding to the chlorosome protein A (CsmA) of Chlorobaculum tepidum, and the aggregated pigment–protein dimer, as determined from protein–ligand docking and quantum chemistry calculations. Our calculations provide strong evidence that the BChl a molecule is coordinated to the His25 residue of CsmA, with the magnesium center of the bacteriochlorin ring situated <3 Å from the imidazole nitrogen atom of the histidine sidechain, and the phytyl tail aligned along the nonpolar residues of the α-helix of CsmA. We also confirm that the Q _y band in the absorption spectra of BChl a experiences a large (+16 to +43 nm) redshift when aggregated with another BChl a molecule in the CsmA dimer, compared to the BChl a in solvent; this redshift has been previously established by experimental researchers. We propose that our model of the BChl a–CsmA binding motif, where the dimer contains parallel aligned N-terminal regions, serves as the smallest repeating unit in a larger model of the para-crystalline chlorosome baseplate protein.     

Photoinduced transient absorbance spectra of P840/P840(+) and the FMO protein in reaction centers of Chlorobium vibrioforme.

The kinetics of photoinduced absorbance changes in the 400-ns to 100-ms time range were studied between 770 and 1025 nm in reaction center core (RCC) complexes isolated from the green sulfur bacterium Chlorobium vibrioforme. A global, multiple stretched-exponential analysis shows the presence of two distinct but strongly overlapping spectra. The spectrum of the 70-micros component consists of a broad bleaching with two minima at 810 and 825 nm and a broad positive band at wavelengths greater than 865 nm and is assigned to the decay of (3)Bchl a of the Fenna-Matthews-Olson (FMO) protein. The contribution of the 70-micros component correlates with the amount of FMO protein in the isolated RCC complex. The spectrum of the 1.6-micros component has a sharp bleaching at 835 nm, a maximum at 805 nm, a broad positive band at wavelengths higher than 865 nm, and a broad negative band at wavelengths higher than 960 nm. When the RCC is incubated with inorganic iron and sulfur, the 1.6-micros component is replaced by a component with a lifetime of approximately 40 micros, consistent with the reconstruction of the F(X) cluster. We propose that the 1.6-micros component results from charge recombination between P840(+) and an intermediate electron acceptor operating between A(0) and F(X). Our studies in Chlorobium RCCs show that approaches that employ a single wavelength in the measurement of absorption changes have inherent limitations and that a global kinetic analysis at multiple wavelengths in the near-infrared is required to reliably separate absorption changes due to P840/P840(+) from the decay of (3)Bchl a in the FMO protein.     

Molecular contacts for chlorosome envelope proteins revealed by cross-linking studies with chlorosomes from Chlorobium tepidum

Chlorosomes are unique light-harvesting antennae found in two phyla of green bacteria: Chlorobi and Chloroflexi. In the green sulfur bacterium Chlorobium tepidum, 10 proteins (CsmA, CsmB, CsmC, CsmD, CsmE, CsmF, CsmH, CsmI, CsmJ, and CsmX) exist in the chlorosome envelope. Chlorosomes from the wild type and mutants lacking a single chlorosome protein were cross-linked with the zero-length cross-linker 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC) and analyzed by gel electrophoresis. Similar cross-linking products were observed when the time and temperature were varied or when EDC was replaced with glutaraldehyde. Specific interactions between chlorosome proteins in cross-linked products were identified by immunoblotting with polyclonal antibodies raised against recombinant chlorosome proteins. We confirmed these interactions by demonstrating that these products were missing in appropriate mutants. Confirming the location of CsmA in the paracrystalline baseplate, cross-linking showed that CsmA forms dimers, trimers, and homomultimers as large as dodecamers and that CsmA directly interacts with the Fenna-Matthews-Olson protein. Cross-linking further suggests that the precursor form of CsmA is inserted near the edges of the baseplate, where CsmA and pre-CsmA interact with CsmB and CsmF. Several chlorosome proteins, including CsmA, CsmC, CsmD, CsmH, CsmI, CsmJ, and CsmX, were shown to exist as homomultimers in the chlorosome envelope. On the basis of the structural information obtained from these cross-linking experiments, a model for the locations and interactions of the proteins of the chlorosome envelope is proposed.     

A model of the protein–pigment baseplate complex in chlorosomes of photosynthetic green bacteria

In contrast to photosynthetic reaction centers, which share the same structural architecture, more variety is found in the light-harvesting antenna systems of phototrophic organisms. The largest antenna system described, so far, is the chlorosome found in anoxygenic green bacteria, as well as in a recently discovered aerobic phototroph. Chlorosomes are the only antenna system, in which the major light-harvesting pigments are organized in self-assembled supramolecular aggregates rather than on protein scaffolds. This unique feature is believed to explain why some green bacteria are able to carry out photosynthesis at very low light intensities. Encasing the chlorosome pigments is a protein-lipid monolayer including an additional antenna complex: the baseplate, a two-dimensional paracrystalline structure containing the chlorosome protein CsmA and bacteriochlorophyll a (BChl a). In this article, we review current knowledge of the baseplate antenna complex, which physically and functionally connects the chlorosome pigments to the reaction centers via the Fenna–Matthews–Olson protein, with special emphasis on the well-studied green sulfur bacterium Chlorobaculum tepidum (previously Chlorobium tepidum). A possible role for the baseplate in the biogenesis of chlorosomes is discussed. In the final part, we present a structural model of the baseplate through combination of a recent NMR structure of CsmA and simulation of circular dichroism and optical spectra for the CsmA–BChl a complex.     

Nine mutants of Chlorobium tepidum each unable to synthesize a different chlorosome protein still assemble functional chlorosomes

Chlorosomes of the green sulfur bacterium Chlorobium tepidum comprise mostly bacteriochlorophyll c (BChl c), small amounts of BChl a, carotenoids, and quinones surrounded by a lipid-protein envelope. These structures contain 10 different protein species (CsmA, CsmB, CsmC, CsmD, CsmE, CsmF, CsmH, CsmI, CsmJ, and CsmX) but contain relatively little total protein compared to other photosynthetic antenna complexes. Except for CsmA, which has been suggested to bind BChl a, the functions of the chlorosome proteins are not known. Nine mutants in which a single csm gene was inactivated were created; these mutants included genes encoding all chlorosome proteins except CsmA. All mutants had BChl c contents similar to that of the wild-type strain and had growth rates indistinguishable from or within approximately 90% (CsmC(-) and CsmJ(-)) of those of the wild-type strain. Chlorosomes isolated from the mutants lacked only the protein whose gene had been inactivated and were generally similar to those from the wild-type strain with respect to size, shape, and BChl c, BChl a, and carotenoid contents. However, chlorosomes from the csmC mutant were about 25% shorter than those from the wild-type strain, and the BChl c absorbance maximum was blue-shifted about 8 nm, indicating that the structure of the BChl c aggregates in these chlorosomes is altered. The results of the present study establish that, except with CsmA, when the known chlorosome proteins are eliminated individually, none of them are essential for the biogenesis, light harvesting, or structural organization of BChl c and BChl a within the chlorosome. These results demonstrate that chlorosomes are remarkably robust structures that can tolerate considerable changes in protein composition.     

The three-dimensional structure of CsmA: a small antenna protein from the green sulfur bacterium Chlorobium tepidum

The structure of the chlorosome baseplate protein CsmA from Chlorobium tepidum in a 1:1 chloroform:methanol solution was determined using liquid-state NMR spectroscopy. The data reveal that the 59-residue protein is predominantly alpha-helical with a long helical domain extending from residues V6 to L36, containing a putative bacteriochlorophyll a binding domain, and a short helix in the C-terminal part extending from residues M41 to G49. These elements are compatible with a model of CsmA having the long N-terminal alpha-helical stretch immersed into the lipid monolayer confining the chlorosome and the short C-terminal helix protruding outwards, thus available for interaction with the Fenna-Matthews-Olson antenna protein.     

Isolation and Characterization of Carotenosomes from a Bacteriochlorophyll c-less Mutant ofChlorobium tepidum

Chlorosomes are the light-harvesting organelles in photosynthetic green bacteria and typically contain large amounts of bacteriochlorophyll (BChl) c in addition to smaller amounts of BChl a, carotenoids, and several protein species. We have isolated vestigial chlorosomes, denoted carotenosomes, from a BChl c-less, bchK mutant of the green sulfur bacterium Chlorobium tepidum. The physical shape of the carotenosomes (86 ± 17 nm × 66 ± 13 nm × 4.3 ± 0.8 nm on average) was reminiscent of a flattened chlorosome. The carotenosomes contained carotenoids, BChl a, and the proteins CsmA and CsmD in ratios to each other comparable to their ratios in wild-type chlorosomes, but all other chlorosome proteins normally found in wild-type chlorosomes were found only in trace amounts or were not detected. Similar to wild-type chlorosomes, the CsmA protein in the carotenosomes formed oligomers at least up to homo-octamers as shown by chemical cross-linking and immunoblotting. The absorption spectrum of BChl a in the carotenosomes was also indistinguishable from that in wild-type chlorosomes. Energy transfer from the bulk carotenoids to BChl a in carotenosomes was poor. The results indicate that the carotenosomes have an intact baseplate made of remarkably stable oligomeric CsmA–BChl a complexes but are flattened in structure due to the absence of BChl c. Carotenosomes thus provide a valuable material for studying the biogenesis, structure, and function of the photosynthetic antennae in green bacteria.     

Initial characterization of the photosynthetic apparatus of "Candidatus Chlorothrix halophila," a filamentous, anoxygenic photoautotroph

“Candidatus Chlorothrix halophila” is a recently described halophilic, filamentous, anoxygenic photoautotroph (J. A. Klappenbach and B. K. Pierson, Arch. Microbiol. 181:17-25, 2004) that was enriched from the hypersaline microbial mats at Guerrero Negro, Mexico. Analysis of the photosynthetic apparatus by negative staining, spectroscopy, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the photosynthetic apparatus in this organism has similarities to the photosynthetic apparatus in both the Chloroflexi and Chlorobi phyla of green photosynthetic bacteria. The chlorosomes were found to be ellipsoidal and of various sizes, characteristics that are comparable to characteristics of chlorosomes in other species of green photosynthetic bacteria. The absorption spectrum of whole cells was dominated by the chlorosome bacteriochlorophyll c (BChl c) peak at 759 nm, with fluorescence emission at 760 nm. A second fluorescence emission band was observed at 870 nm and was tentatively attributed to a membrane-bound antenna complex. Fluorescence emission spectra obtained at 77 K revealed another complex that fluoresced at 820 nm, which probably resulted from the chlorosome baseplate complex. All of these results suggest that BChl c is present in the chlorosomes of “Ca. Chlorothrix halophila,” that BChl a is present in the baseplate, and that there is a membrane-bound antenna complex. Analysis of the proteins in the chlorosomes revealed an ~6-kDa band, which was found to be related to the BChl c binding protein CsmA found in other green bacteria. Overall, the absorbance and fluorescence spectra of “Ca. Chlorothrix halophila” revealed an interesting mixture of photosynthetic characteristics that seemed to have properties similar to properties of both phyla of green bacteria when they were compared to the photosynthetic characteristics of Chlorobium tepidum and Chloroflexus aurantiacus.     

Association of bacteriochlorophyll a with the CsmA protein in chlorosomes of the photosynthetic green filamentous bacterium Chloroflexus aurantiacus.

The protein assumed to be associated with bacteriochlorophyll (BChl) a in chlorosomes from the photosynthetic green filamentous bacterium Chloroflexus aurantiacus was investigated by alkaline treatment, proteolytic digestion and a new treatment using 1-hexanol, sodium cholate and Triton X-100. Upon alkaline treatment, only the 5.7 kDa CsmA protein was removed from the chlorosomes among six proteins detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis, concomitantly with the disappearance of BChl a absorption at 795 nm. Trypsin treatment removed two proteins with molecular masses of 11 and 18 kDa (CsmN and CmsM), whereas the spectral properties of BChl a and BChl c were not changed. By the new hexanol-detergent (HD) treatment, most BChl c and all of the detected proteins except CsmA were removed from the chlorosomes without changing the BChl a spectral properties. Subsequent proteinase K treatment of these HD-treated chlorosomes caused digestion of CsmA and a simultaneous decrease of the BChl a absorption band. Based on these results, we suggest that CsmA is associated with BChl a in the chlorosomes. This suggestion was supported by the measured stoichiometric ratio of BChl a to CsmA in isolated chlorosomes, which was estimated to be between 1.2 and 2.7 by amino acid analysis of the SDS-PAGE-resolved protein bands.     

Distinctive properties of bacilliform photosynthetic heliobacteria

Abstract Heliobacterium chlorum and Heliobacillus mobilis are closely related N₂-fixing anoxyphoto-trophs that contain bacteriochlorophyll g (Bchl g ) as the major photopigment. In the presence of O₂ and light, the absorbance peak in the infra-red (788 nm) of this novel photoreceptor disappears and absorbance at 670 nm increases simultaneously. These optical changes appear to be due to a photoisomerization reaction which converts Bchl g to a form of green-plant chlorophyll a (in which farnesol replaces phytol). In addition to this unusual property, the Gram-negative heliobacteria present biochemical features (16S RNA base sequence and peptidoglycan structure) indicating an evolutionary relationship with some Gram-positive bacteria. In comparison to H. chlorum, H. mobilis grows more robustly and shows a much lower tendency to spheroplast and lyse; accordingly, H. mobilis is better suited for further investigations on the biology and biochemistry of these exceptional prokaryotes.     

On the influence of pigment-protein interactions on the energy transfer processes in photosynthetic membrane structures. 2. LH2 complex of Rhodobacter sphaeroides

Low-temperature heterogeneous absorption and circular dichroism spectra of the Rb. sphaeroides LH2 complexes are calculated within the framework of the mini-exciton theory and diagonal static random disorder for the pure electronic transitions of the monomeric Bchl molecules. The coupling of Bchl molecules with the surrounding amino acid residues has been shown to change both the exciton distribution between the pigment molecules in each of the exciton states. The value of the delocalization index depends on the excitation wavelength and varies between 2-6 Bchl molecules. The optical transitions occurring at 780-790 and 820 nm have been found to be strongly mixed so that all Bchl molecules of the LH2 complex predetermine absorption in these spectral regions. On the other hand, absorption at 800 and 850 nm is mainly determined by the cycles of 9 and 18 Bchl molecules, respectively. Thus, the light energy absorbed by the B800 molecules at 800 nm is transferred to the B850 molecules by the interlevel exciton relaxation processes due to the population of the heavily mixed 820-nm exciton levels. The width of the heterogeneous absorption band for the cyclic monomeric aggregate has been shown to decrease as compared with the monomeric absorption band by square root(Ndel) time, where Ndel is the mean number of pigments over which the exciton is delocalized within the excited absorption band.     

Differentiation of the intracytoplasmic membrane of Rhodopseudomonas palustris induced by variations of oxygen partial pressure or light intensity

The photosynthetic apparatus of Rhodopseudomonas palustris contains, in addition to reaction center bacteriochlorophyll (Bchl) two spectral forms of light harvesting (LH) Bchl, i.e. LH Bchl I, characterized by an infrared absorption maximum at 880 nm (890 nm at 77°K) and LH Bchl II absorbing at 805 and 855 nm (805 and 870 nm at 77°K). LH Bchl I seems to be associated with a single protein species of an apparent mol. wt. of 13000 whereas LH Bchl II is apparently associated with two proteins of mol. wts. of 9000 and 11000.Cells in anaerobic cultures adapt to changes of light intensity 1. by variation of the size of the photosynthetic unit, i.e. the molar ratio of LH Bchl II to reaction center Bchl, 2. by variation of the number of photosynthetic units per unit of membrane area, 3. by regulation of the size of the intracytoplasmic membrane system.During adaptation of changes of oxygen partial pressure cells are able to synthesize reaction center Bchl, LH Bchl and intracytoplasmic membranes at different rates. The synthesis of reaction center Bchl and LH Bchl I are, however, coordinated with each other, while the syntheses of LH Bchl II and reaction center Bchl proceed independently.List of Non-Standard Abbreviations Bchl bacteriochlorophyll - ICM mitracytoplasmic membrane - LDAO lauryldimethyl aminoxide - R Rhodopseudomonas - RC reaction center - SDS sodium dodecylsulfate     

Isolation and characterization of cytoplasmic membranes and chlorosomes from the green bacterium Chloroflexus aurantiacus.

A method was developed which allows the isolation and purification of cytoplasmic membranes and chlorosomes from cells of Chloroflexus aurantiacus grown under different light conditions. The dipolar ionic detergent Deriphat (0.08%) and a sodium iodide gradient centrifugation were used in isolating cytoplasmic membranes. Chlorosomes were prepared with 0.16% of the dipolar ionic detergent Miranol and purified by a sucrose gradient centrifugation. Cytoplasmic membrane fractions prepared from either high- (3,000 W m-2), medium-(200 W m-2) or low- (7 W m-2) light-grown cells had near infrared absorption bands at 866, 808, and 755 nm in a constant characteristic absorbance ratio of 6:3.8:1. In all cytoplasmic membrane preparations, the amount of bacteriochlorophyll a (Bchl a) per cytochrome, the amount of Bchl a per reaction center, and reaction center per milligram of cytoplasmic membrane protein was found to be constant. No Bchl c was present. Five respiratory enzyme activities have been measured in the cytoplasmic membrane fraction. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of denatured cytoplasmic membrane showed many bands, but a major polypeptide with an apparent molecular weight of 8,000. In contrast, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified chlorosomes did not contain the 8,000-molecular-weight band but revealed only three distinct protein bands with molecular weights of 15,000, 12,000, and 6,000. Isolated chlorosomes contained Bchl c and a small, yet constant, amount of Bchl a (absorbing at 790 nm) in a molar ratio of 25:1. The data indicated that the components of the photosynthetic apparatus in the cytoplasmic membrane of Chloroflexus aurantiacus remained constant and only the amount of antenna Bchl c varied with light conditions.     

The chlorosome: a prototype for efficient light harvesting in photosynthesis

Three phyla of bacteria include phototrophs that contain unique antenna systems, chlorosomes, as the principal light-harvesting apparatus. Chlorosomes are the largest known supramolecular antenna systems and contain hundreds of thousands of BChl c/d/e molecules enclosed by a single membrane leaflet and a baseplate. The BChl pigments are organized via self-assembly and do not require proteins to provide a scaffold for efficient light harvesting. Their excitation energy flows via a small protein, CsmA embedded in the baseplate to the photosynthetic reaction centres. Chlorosomes allow for photosynthesis at very low light intensities by ultra-rapid transfer of excitations to reaction centres and enable organisms with chlorosomes to live at extraordinarily low light intensities under which no other phototrophic organisms can grow. This article reviews several aspects of chlorosomes: the supramolecular and molecular organizations and the light-harvesting and spectroscopic properties. In addition, it provides some novel information about the organization of the baseplate.     

A PM6 study of <Emphasis Type="Italic">Rhodopseudomonas Acidophila</Emphasis> light harvesting center II B800 bacteriochlorophylls in representative protein environment

Bacterial light-harvesting II (LH-II) centers contain two types of Bacteriochlorophylls (Bchl). One is named B800 and found as a single molecule within one monomer of the complex while the other named B850 is found as a dimer. Their names indicate their peak of UV absorbance around red spectrum. Both types of molecules are attached to the protein chain via ligation of their central Magnesium atom to an either Histidine or Deoxymethionine amino acid. They are also coordinated by peripheral hydrogen bonds that they accept with their carboxyl side group. Both the ligation and the hydrogen bonding are thought to have an effect on electronic structure of the Bchl hence its UV absorbance and energy transfer rate. Experiments and theoretic studies performed on this subject support the above idea. This theoretical molecular modeling study case aims to mimic the experimental mutations performed on certain amino acids in silico and study its effects on the electronic structure of Bchl. By comparison with experimental results it was observed that the likely place for the nearby Arginine is not below the plane of the Bchl as in the X-ray crystallographic structure but above the plane defined by the four nitrogen atoms and their rings. It was also seen that the coordination of the acetyl group is very sensitive to changes in ligation of the Bchl molecule.     

X-ray scattering and electron cryomicroscopy study on the effect of carotenoid biosynthesis to the structure of Chlorobium tepidum chlorosomes

Chlorosomes, the main antenna complexes of green photosynthetic bacteria, were isolated from null mutants of Chlorobium tepidum, each of which lacked one enzyme involved in the biosynthesis of carotenoids. The effects of the altered carotenoid composition on the structure of the chlorosomes were studied by means of x-ray scattering and electron cryomicroscopy. The chlorosomes from each mutant strain exhibited a lamellar arrangement of the bacteriochlorophyll c aggregates, which are the major constituents of the chlorosome interior. However, the carotenoid content and composition had a pronounced effect on chlorosome biogenesis and structure. The results indicate that carotenoids with a sufficiently long conjugated system are important for the biogenesis of the chlorosome baseplate. Defects in the baseplate structure affected the shape of the chlorosomes and were correlated with differences in the arrangement of lamellae and spacing between the lamellar planes of bacteriochlorophyll aggregates. In addition, comparisons among the various mutants enabled refinement of the assignments of the x-ray scattering peaks. While the main scattering peaks come from the lamellar structure of bacteriochlorophyll c aggregates, some minor peaks may originate from the paracrystalline arrangement of CsmA in the baseplate.     

Molecular characterization of novel red green nonsulfur bacteria from five distinct hot spring communities in Yellowstone National Park.

We characterized and compared five geographically isolated hot springs with distinct red-layer communities in Yellowstone National Park. Individual red-layer communities were observed to thrive in temperatures ranging from 35 to 60 degrees C and at pH 7 to 9. All communities were dominated by red filamentous bacteria and contained bacteriochlorophyll a (Bchl a), suggesting that they represented novel green nonsulfur (GNS) bacteria. The in vivo absorption spectra of individual sites were different, with two sites showing unusual Bchl a protein absorption bands beyond 900 nm. We prepared and analyzed 16S rRNA libraries from all of these sites by using a combination of general bacterial primers and new GNS-specific primers described here. These studies confirmed the presence of novel GNS-like bacteria in all five communities. All GNS-like clones were most similar to Roseiflexus castenholzii, a red filamentous bacterium from Japan that also contains only Bchl a. Phylogenies constructed by using GNS-like clones from Yellowstone red-layer communities suggest the presence of a moderately diverse new "red" cluster within the GNS lineage. Within this cluster, at least two well-supported subclusters emerged: YRL-A was most similar to Roseiflexus and YRL-B appeared to be novel, containing no known isolates. While these patterns showed some site specificity, they did not correlate with observed Bchl a spectrum differences or obvious features of the habitat.